The present invention relates to a novel protein having a cytokinin-type activity, to the genetic engineering tools for its production, namely a recombinant DNA, an expression vector carrying this recombinant DNA, and the procaryotic microorganisms and the eucaryotic cells containing this recombinant DNA, and to a drug, useful especially as an anticancer agent or immunomodulator, in which this protein is present as the active principle.
It is well known that the immune system comprises cellular elements and soluble substances, called cytokinins, secreted by said elements. Cytokinins are proteins which effect communication between an emitter cell and a target cell belonging either to the immune system or to another biological system of the organism. In general, cytokinins have a so-called pleiotropic biological activity, i.e. they can have multiple effects on the target cell: proliferation, differentiation, cytolysis, activation, chemotaxis etc. Several of these molecules have already found applications in therapeutics: for example, interleukin-2 or interferon-.alpha. used for the treatment of certain tumors by immuno-therapy, and myelopoietic factors, such as GCSF (Granulocyte Colony Stimulating Factor) or GMCSF (Granulocyte Monocyte Colony Stimulating Factor), which stimulate the growth and differentiation of blood cells and whereby blood which has been impoverished in blood cells as a result of chemotherapy can be enriched therewith.
One of the first cytokinins to be discovered was interleukin-1, to which a central activity in inflammation--chemotaxis of neutrophils--was initially attributed following experiments showing an activity of this type in vivo after injection (J. Oppenheim et al., 1986, Imm. Today, 7, 45-56). It is now known that interleukin-1 stimulates in vivo the expression of another cytokinin which is chemotactic towards neutrophils, namely interleukin-8 (originally called Neutrophil Chemotactic Factor, NCF). This cytokinin, whose amino acid sequence was determined in 1987 after isolation and purification and also by cloning and sequencing of its complementary DNA (K. Matsushima et al., 1988, J. Exp. Med., 1883-1893), is homologous with other cytokinins already known at the time of its discovery, namely the cytokinins produced by the .alpha. granules of platelets, such as PF4 (Platelet Factor 4) and PBP (Platelet Basic Protein). The family of known proteins homologous with interleukin-8, usually called the SIS family (representing Small Induced Secreted proteins), has grown considerably since 1987 [J. Oppenheim et al., 1991, Ann. Rev. Immun., 9, 617]. It currently includes the following cytokinins in particular: gro (also called MGSA: Melanoma Growth Stimulatory Activity) described by A. Anisowicz et al., 1987, Proc. Ntl. Acad. Sci. U.S.A., 84, 7188-7192, and A. Richmond et al., 1988, EMBO J., 7, 2025-2033, RANTES (Regulated upon Activation Normal T Expressed and presumably Secreted) described by T. Schall et al., 1988, J. Imm., 141, 1018-1025, MIP-1 (Macrophage Inflammatory Protein 1) described by S. D. Wolpe et al., 1988, J. Exp. Med., 167, 570-581, MIP-2 (Macrophage Inflammatory Protein 2) described by S. D. Wolpe et al., 1989, Proc. Ntl. Acad. Sci. U.S.A., 86, 612-616, and MCP-1 (Monocyte Chemoattractant Protein 1, also called MCAF: Monocyte Chemotactic and Activating Factor) described by Yoshimura et al., 1989, Proc. Ntl. Acad. Sci. U.S.A., 84, 9233-9237, and K. Matsushima et al., 1989, J. Expr. Med., 169, 1485-1489.
The cytokinin MCP-1, isolated from a line of gliomas by T. Yoshimura, op. cit., and from a line of monocytes by K. Matsushima et al., op. cit., exists in two forms with apparent molecular weights of 13 and 15 kDa, called MCP-1.alpha. and MCP-1.beta., which seem to correspond to post-translational modifications [Y. Jiang et al., 1990, J. Biol. Chem., 265, 1318-321]. The cytokinin MCP-1 has a chemotactic activity towards monocytes and basophils but not towards neutrophils (E. J. Leonard, 1990, Immunology Today, 11, 3, 97-101) and a stimulating effect on the cytostatic activity of monocytes on certain tumoral lines (K. Matsushima et al., 1989, J. Exp. Med., 169, 1485-1490).
Three-dimensional structural studies on interleukin-8 and PF4 by nuclear magnetic resonance spectroscopy or X-ray diffraction have shown that these two cytokinins have the same conformation, with a carboxy-terminal peptide of 12 to 15 amino acids in the form of an .alpha. helix (R. St. Charles et al., 1988, J. Biol. Chem., 264, 4, 2092-2099, and G. M. Clore, 1990, Biochem., 29, 1689-1696). According to G. M. Clore, the majority of cytokinins in the SIS family have such a carboxy-terminal part in the form of an a helix, the role of this helix being as yet undetermined [C. Herbert et al., 1991, J. Biol. Chem., 266, 18989-994].
It has recently been shown that the carboxy-terminal peptide of 13 amino acids can have the anti-angiogenic activity (inhibition of the proliferation of blood vessels) of the cytokinin PF4 (T. Maione et al., 1990, Science, 247, 77-79). According to D. G. Osterman, 1982, Biochem. Biophys. Res. Commun., 107, 130-135, this peptide has a chemotactic activity towards monocytes which is thirty times greater than that of PF4.